The present invention relates to methods for displaying (poly)peptides/proteins on the surface of bacteriophage particles by attaching the (poly)peptide/proteins via disulfide bonds. A number of documents are cited throughout this specification. The disclosure content of these documents is herewith incorporated by reference in their entirety.
Smith first demonstrated in 1985 that filamentous phage tolerate foreign protein fragments inserted in their gene III protein (pIII), and could show that the protein fragments are presented on the phage surface (Smith, 1985). Ladner extended that concept to the screening of repertoires of (poly)peptides and/or proteins displayed on the surface of phage (WO 88/06630; WO 90/02809) and, since then, phage display has experienced a dramatic progress and resulted in substantial achievements.
Various formats have been developed to construct and screen (poly)peptide/protein phage-display libraries, and a large number of review articles and monographs cover and summarise these developments (e.g., Kay et al., 1996; Dunn, 1996; McGregor, 1996).
Most often, filamentous phage-based systems have been used.
Initially proposed as display of single-chain Fv (scFv) fragments (WO 88/06630; see additionally WO 92/01047), the method has rapidly been expanded to the display of bovine pancreatic trypsin inhibitor (BPTI) (WO 90/02809), peptide libraries (WO 91/19818), human growth hormone (WO 92/09690), and of various other proteins including the display of multimeric proteins such as Fab fragments (WO 91/17271; WO 92/01047).
To anchor the peptide or protein to the filamentous bacteriophage surface, mostly genetic fusions to phage coat proteins are employed. Preferred are fusions to gene III protein (Parmley and Smith, 1988) or fragments thereof (Bass et al., 1990), and gene VIII protein (Greenwood et al., 1991). In one case, gene VI has been used (Jespers et al., 1995), and recently, a combination of gene VII and gene IX has been used for the display of Fv fragments (Gao et al., 1999).
Furthermore, phage display has also been achieved on phage lambda. In that case, gene V protein (Maruyama et al., 1994), gene J protein, and gene D protein (Sternberg and Hoess, 1995; Mikawa et al., 1996) have been used.
Besides using genetic fusions, foreign peptides or proteins have been attached to phage surfaces via association domains. In WO 91/17271, it was suggested to use a tag displayed on phage and a tag binding ligand fused to the peptide/protein to be displayed to achieve a non-covalent display.
A similar concept was pursued for the display of cDNA libraries (Crameri and Suter, 1993). There the jun/fos interaction was used to mediate the display of cDNA fragments. In their construct, additional cysteine residues flanking both ends of jun as well as fos further stabilised the interaction by forming two disulfide bonds
When screening phage display libraries in biopanning the problem remains how best to recover phage which have bound to the desired target. Normally, this is achieved by elution with appropriate buffers, either by using a pH- or salt gradient, or by specific elution using soluble target. However, the most interesting binders which bind with high affinity to the target might be lost by that approach. Several alternative methods have been devised which try to overcome that problem, either by providing a cleavage signal between the (poly)peptide/protein being displayed and its fusion partner, or between the target of interest and its carrier which anchors the target to a solid surface.
Furthermore, all the approaches referred to hereinabove require to use fusion proteins comprising at least part of a phage coat protein and a foreign (poly)peptide/protein. Especially in the case of using gene III as partner for peptides/proteins to be displayed, this leads to several problems. First, the expression product of gene III is toxic to the host cell, which requires tight regulation of gene III fusion proteins. Second, expression of gene III products can make host cells resistant to infection with helper phage required for the production of progeny phage particles. And finally, recombination events between gene III fusion constructs and wild type copies of gene III lead to undesired artefacts. Furthermore, since at least the C-terminal domain of the gene III protein comprising about 190 amino acids has to be used in order to achieve incorporation of the fusion protein into the phage coat, the size of the vectors comprising the nucleic acid sequences is rather larger, leading to a decrease in transformation efficiency. Transformation efficiency, however, is a crucial factor for the production of very large libraries. Additionally, for the characterisation of (poly)peptide/proteins obtained after selection from a phage display library, the (poly)peptide/protein are usually recloned into expression vectors in order to remove the phage coat protein fusion partner, or in order to create new fusion proteins such as by fusion to enzymes for detection or to multimerisation domains. It would be advantageously to have a system which would allow direct expression without recloning, and direct coupling of the (poly)peptide/protein to other moieties.
Furthermore, most of these approaches (except for the work of Jespers et al. (1995), WO 91/17271, and Crameri and Suter (1993) mentioned hereinabove) are limited to the presentation of (poly)peptides/proteins having a free N-terminus, since the (poly)peptides/proteins have to be fused at the C-terminus with a phage coat protein. Especially in the case of cDNA libraries, or in the case of proteins requiring a free C-terminus to be functional, it would be highly desirable to have a simple method which doesn""t require the generation of C-terminal fusions.
Thus, the technical problem underlying the present invention is to develop a simple, reliable system which enables the presentation of (poly)peptides/proteins on phage particles without the need to use fusion proteins with phage coat proteins. Additionally, there is a need for a method which allows to recover tightly binding (poly)peptides/proteins in a more reliable way.
The solution to this technical problem is achieved by providing the embodiments characterised in the claims. Accordingly, the present invention allows to easily create and screen large libraries of (poly)peptides/proteins displayed on the surface of bacteriophage particles. The technical approach of the present invention, i.e. linking (poly)peptides/proteins by disulfide bonds to the surface of phage particles, is neither provided nor suggested by the prior art.
Thus, the present invention relates to a method for displaying a (poly)peptide/protein on the surface of a bacteriophage particle comprising:
causing or allowing the attachment of said (poly)peptide/protein after expression to a member of the protein coat of said bacteriophage particle, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in said member of the protein coat.
In the context of the present invention, the term xe2x80x9cbacteriophagexe2x80x9d relates to bacterial viruses forming packages consisting of a protein coat containing nucleic acid required for the replication of the phages. The nucleic acid may be DNA or RNA, either double or single stranded, linear or circular. Bacteriophage such as phage lambda or filamentous phage (such as M13, fd, or fl) are well known to the artisan of ordinary skill in the art. In the context of the present invention, the term xe2x80x9cbacteriophage particlesxe2x80x9d refers to the particles according to the present invention, i.e. to particles displaying a (poly)peptide/protein via a disulfide bonds. During the assembly of bacteriophages, the coat proteins may package different nucleic acid sequences, provided that they comprise a packaging signal. In the context of the present invention, the term xe2x80x9cnucleic acid sequencesxe2x80x9d contained in bacteriophages or bacteriophage particles relates to nucleic acid sequences or vectors having the ability to be packaged by bacteriophage coat proteins during assembly of bacteriophages or bacteriophage particles. Preferably said nucleic acid sequences or vectors are derived from naturally occurring genomes of bacteriophage, and comprise for example, in the case of filamentous phage, phage and phagemid vectors. The latter are plasmids containing a packaging signal and a phage origin of replication in addition to plasmid features.
The term xe2x80x9c(poly)peptidexe2x80x9d relates to molecules consisting of one or more chains of multiple, i.e. two or more, amino acids linked via peptide bonds.
The term xe2x80x9cproteinxe2x80x9d refers to (poly)peptides where at least part of the (poly)peptide has or is able to acquire a defined three-dimensional arrangement by forming secondary, tertiary, or quaternary structures within and/or between its (poly)peptide chain(s). This definition comprises proteins such as naturally occurring or at least partially artificial proteins, as well as fragments or domains of whole proteins, as long as these fragments or domains are able to acquire a defined three-dimensional arrangement as described above.
Examples of (poly)peptides/proteins consisting of one chain are single-chain Fv antibody fragments, and examples for (poly)peptides/proteins consisting of more chains are Fab antibody fragments.
When the first cysteine residue is located at the C-terminus of the (poly)peptide/protein, the display format corresponds to the conventional display set-up with the C-terminus being genetically fused to the member of the phage coat protein. However, by using the N-terminus of the (poly)peptide/protein, the display format can be reverted as in the pJuFO system of Crameri and Suter referred to above.
The term xe2x80x9csurface of a bacteriophage particlexe2x80x9d refers to the part of a bacteriophage particle which is in contact with the medium the particle is contained in and which is accessible. The surface is determined by the proteins being part of the phage coat (the members of the protein coat of the particle) which is assembled during phage production in appropriate host cells.
The term xe2x80x9cafter expressionxe2x80x9d refers to the situation that nucleic acid encoding said (poly)peptide/protein is expressed in a host cell prior to attachment of the (poly)peptide/protein to said coat, in contrast to approaches where nucleic acid encoding fusion proteins with bacteriophage coat proteins are being expressed. The expression of nucleic acid encoding said (poly)peptide/protein and the step of causing or allowing the attachment may be performed in separated steps and/or environments. Preferably, however, expression and the step of causing or allowing the attachment are being performed sequentially in an appropriate host cell. The term xe2x80x9cwherein said attachment is caused by the formation of a disulfide bondxe2x80x9d refers to a situation, wherein the disulfide bond is responsible for the attachment, and wherein no interaction domain for interaction with a second domain present in the (poly)peptide/protein has been recombinantly fused to said member of the protein coat, as for example in the case of the pJuFo system (Crameri and Suter, 1993).
In a preferred embodiment, the bacteriophage particle displaying the (poly)peptide/protein contains a nucleic acid sequence encoding the (poly)peptide/protein.
Methods for construction of nucleic acid molecules encoding a (poly)peptide/protein according to the present invention, for construction of vectors comprising said nucleic acid molecules, for introduction of said vectors into appropriately chosen host cells, for causing or allowing the expression of said (poly)peptides/proteins are well-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1999; Ge et al, 1995). Further well-known are methods for the introduction of genetic material required for the generation of progeny bacteriophages or bacteriophage particles in appropriate host cells, and for causing or allowing the generation of said progeny bacteriophages or bacteriophage particles (see, e.g., Kay et al., 1996).
In a further preferred embodiment, the present invention relates to a method, wherein said second cysteine residue is present at a corresponding amino acid position in a wild type coat protein of a bacteriophage.
In a yet further preferred embodiment, the present invention relates to a method, wherein said member of the protein coat is a wild type coat protein of a bacteriophage.
The term xe2x80x9cwild type coat proteinxe2x80x9d refers to those proteins forming the phage coat of naturally occurring bacteriophages. In the case of filamentous bacteriophage, said wild type proteins are gene III protein (pIII), gene VI protein (pVI), gene VII protein (pVII), gene VIII protein (pVIII), and gene IX protein (pIX). The sequences, including the differences between the closely related members of the filamentous bacteriophages such as f1, fd, and M13, are well known to one of ordinary skill in the art (see, e.g., Kay et al., 1996).
In a further preferred embodiment, said member of the protein coat is a truncated variant of a wild type coat protein of a bacteriophage, wherein said truncated variant comprises at least that part of said wild type coat protein causing the incorporation of said coat protein into the protein coat of the bacteriophage particle.
The term xe2x80x9ctruncated variantxe2x80x9d refers to proteins derived from the wild type proteins referred to above which are modified by deletion of at least part of the wild type sequences. This comprises variants such as truncated gene III protein variants which have been found in bacteriophage mutants (Crissman and Smith, 1984) or which have been generated in the course of standard phage display methods (e.g. Bass et al., 1990; Krebber, 1996). For example, said truncated variant may consist, or include, the C-terminal domain of the gene III protein. To identify truncated variants according to the present invention, a detection tag may be fused to the variant, and an assay may be set up to determine whether the variant is incorporated into the phage coat of bacteriophage particles formed in the presence of the variant. By way of truncating a wild type protein by deleting a part of the wild type protein, a cysteine residue may become available which in the wild type protein was forming a disulfide bond with a second cysteine comprised in the deleted part.
In a yet further preferred embodiment, said member of the protein coat is a modified variant of a wild type coat protein of a bacteriophage, wherein said modified variant is capable of being incorporated into the protein coat of the bacteriophage particle.
Methods for achieving modification of a wild type protein according to the present invention are well-known to one of ordinary skill in the art, and involve standard cloning and/or mutagenesis techniques. Methods for the construction of nucleic acid molecules encoding a modified variant of a wild type protein used in a method according to the present invention, for construction of vectors comprising said nucleic acid molecules, including the construction of phage and/or phagemid vectors, for introduction of said vectors into appropriately chosen host cells, for causing or allowing the expression of said modified protein are well-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1999; Kay et al., 1996). To identify modified variants according to the present invention, a detection tag may be fused to the variant, and an assay may be set up to determine whether the variant is capable or being incorporated into the phage coat of bacteriophage particles formed in the presence of the variant.
In a most preferred embodiment, said second cysteine residue is not present at a corresponding amino acid position in a wild type coat protein of a bacteriophage.
In a preferred embodiment, said second cysteine has been artificially introduced into a wild type coat protein of a bacteriophage.
In the context of the present invention, the term xe2x80x9cartificially introducedxe2x80x9d refers to a situation where a wild type coat protein has been modified by e.g. recombinant means. For example, nucleic acid encoding a wild type coat protein may be manipulated by standard procedures to introduce a cysteine codon creating a nucleic acid sequence encoding a modified coat protein, wherein a cysteine residue is artificially introduced by insertion into, or addition of said cysteine residue to, said at least part of a wild type or modified coat protein, or by substitution of an amino acid residue comprised in said at least part of a wild type or modified protein by said cysteine residue, or by fusion of said at least part of a wild type or modified coat protein with a (poly)peptide/protein comprising said second cysteine residue, or by any combination of said insertions, additions, substitutions or fusions. Upon expression of the nucleic acid comprising such recombinantly introduced cysteine codon, a variant of the wild type protein is formed comprising a cysteine residue.
In a further most preferred embodiment, said second cysteine has been artificially introduced into a truncated variant of a wild type coat protein of a bacteriophage.
In a yet further preferred embodiment, said second cysteine has been artificially introduced into a modified variant of a wild type coat protein of a bacteriophage.
Methods for achieving the artificial introduction according to the present invention are well-known to one of ordinary skill in the art, and involve standard cloning and/or mutagenesis techniques. Methods for the construction of nucleic acid molecules encoding a modified variant of a wild type protein used in a method according to the present invention, for construction of vectors comprising said nucleic acid molecules, for introduction of said vectors into appropriately chosen host cells, for causing or achieving the expression of said fusion proteins are well-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1999).
In another embodiment, the present invention relates to a method, wherein said second cysteine is present at, or in the vicinity of, the C-or the N-terminus of said member of the phage coat of said bacteriophage particle.
The term xe2x80x9cin the vicinity ofxe2x80x9d refers to a stretch of up to 15, or more preferably, up to 10 amino acids, counted in both cases from either N-or C-terminus of said (poly)peptide/protein, provided that the N-or C-terminus is located at the outside of the bacteriophage.
Yet further preferred is a method, wherein said bacteriophage is a filamentous bacteriophage. Filamentous bacteriophage such as M13, fd, or f1 are well known to the artisan of ordinary skill in the art.
In the case of filamentous bacteriophage, a method is particularly preferred, wherein said member of the protein coat of the bacteriophage particle is or is derived from the wild type coat protein pIII.
Further preferred is a method, wherein said member of the protein coat of the bacteriophage particle is or is derived from the wild type coat protein pIX. In the context of the present invention, the term xe2x80x9cis derivedxe2x80x9d refers to a modification, wherein the modified protein is capable of being incorporated into the protein coat of the bacteriophage particle. Preferably, those parts of the modified protein corresponding to the wild type protein exhibit an amino acid identity exceeding about 70%, preferably about 80%, most preferably about 90% compared to the corresponding wild type sequence.
In a yet further preferred embodiment of the present invention, the method comprises:
(a) providing a host cell harbouring a nucleic acid sequence comprising a nucleic acid sequence encoding said (poly)peptide/protein;
(b) causing or allowing the expression of said nucleic acid sequence; and
(c) causing or allowing the production of bacteriophage particles in said host cell.
In the context of the present invention, the term xe2x80x9ccausing or allowing the expressionxe2x80x9d describes cultivating host cells under conditions such that nucleic acid sequence is expressed.
Methods for construction of nucleic acid molecules encoding a (poly)peptide/protein according to to the present invention, for construction of vectors comprising said nucleic acid molecules, for introduction of said vectors into appropriately chosen host cells, for causing or allowing the expression of (poly)peptides/proteins are well-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1999). Further well-known are methods for the introduction of genetic material required for the generation of progeny bacteriophages or bacteriophage particles in appropriate host cells, and for causing or allowing the generation of said progeny bacteriophages or bacteriophage particles (see, e.g., Kay et al., 1996). The step of causing or allowing the production of bacteriophage particles may require the use of appropriate helper phages, e.g. in the case of working with phagemids.
The steps (b) and (c) may be performed sequentially, in either order, or simultaneously.
In a still further embodiment, said (poly)peptide/protein comprises an immunoglobulin or a functional fragment thereof.
In this context, xe2x80x9cimmunoglobulinxe2x80x9d is used as a synonym for xe2x80x9cantibodyxe2x80x9d. The term xe2x80x9cfunctional fragmentxe2x80x9d refers to a fragment of an immunoglobulin which retains the antigen-binding moiety of an immunoglobulin. Functional immunoglobulin fragments according to the present invention may be Fv (Skerra and Plxc3xcckthun, 1988), scFv (Bird et al., 1988; Huston et al., 1988), disulfide-linked Fv (Glockshuber et al., 1992; Brinkmann et al., 1993), Fab, F(abxe2x80x2)2 fragments or other fragments well-known to the practitioner skilled in the art, which comprise the variable domain of an immunoglobulin or immunoglobulin fragment.
Particularly preferred is an scFv or Fab fragment.
In a preferred embodiment, the present invention relates to a nucleic acid sequence encoding a modified variant of a wild type coat protein of a bacteriophage, wherein said modified variant consists of:
(a) one or more parts of said wild type coat protein of a bacteriophage, wherein one of said parts comprises at least that part which causes or allows the incorporation of said coat protein into the phage coat; and
(b) between one and six additional amino acid residues not present at the corresponding amino acid positions in a wild type coat protein of a bacteriophage, wherein one of said additional amino acid residues is a cysteine residue.
In the context of the present invention, a modified variant obtained by substitution of an amino acid residue in a wild type coat protein sequence by a cysteine residue may be regarded as a variant composed of two parts of said wild type protein linked by an additional cysteine residue. Correspondingly, variants of a wild type coat protein comprising several mutations compared to the wild type sequence may be regarded as being composed of several wild type parts, wherein the individual parts are linked by the mutated residues. However, said variant may also result from the addition of up to six residues, including a cysteine residue, to either C-and or N-terminus of the wild type coat protein.
Further preferred is a nucleic acid sequence encoding a modified variant of a wild type coat protein of a bacteriophage, wherein said modified variant consists of:
(a) one or more parts of said wild type coat protein of a bacteriophage, wherein one of said parts comprises at least that part which causes or allows the incorporation of said coat protein into the phage coat;
(b) between one and six additional amino acid residues not present at the corresponding amino acid positions in a wild type coat protein of a bacteriophage, wherein one of said additional amino acid residues is a cysteine residue; and
(c) one or more peptide sequences for purification and/or detection purposes.
Particularly preferred are peptides comprising at least five histidine residues (Hochuli et at., 1988), which are able to bind to metal ions, and can therefore be used for the purification of the protein to which they are fused (Lindner et al., 1992). Also provided for by the invention are additional moieties such as the commonly used c-myc and FLAG tags (Hopp et al., 1988; Knappik and Plxc3xcckthun, 1994), or the Strep-tag (Schmidt and Skerra, 1994; Schmidt et al., 1996).
The modified variant may further comprise amino acid residues required for cloning, for expression, or protein transport. Amino acid residues required for cloning may include residues encoded by nucleic acid sequences comprising recognition sequences for restriction endonucleases which are incorporated in order to enable the cloning of the nucleic acid sequences into appropriate vectors. Amino acid residues required for expression may include residues leading to increased solubility or stability of the (poly)peptide/protein. Amino acid residues required for protein transport may include signalling sequences responsible for the transport of the modified variant to the periplasm of E. coli, and/or amino acid residues facilitating the efficient cleavage of said signalling sequences. Further amino acid residues required for cloning, expression, protein transport, purification and/or detection purposes referred to above are numerous moieties well known to the practitioner skilled in the art.
In another embodiment, the present invention relates to a vector comprising a nucleic acid sequence according to the present invention.
In a preferred embodiment, the vector further comprises one or more nucleic acid sequences encoding a (poly)peptide/protein comprising a second cysteine residue.
In a most preferred embodiment, said (poly)peptide/protein comprises an immunoglobulin or a functional fragment thereof.
In the case of single-chain Fv antibody fragments referred to hereinabove, the vector comprises one nucleic acid sequence encoding the VH and VL domains linked by a (poly)peptide linker, and in the case of Fab antibody fragments, the vector comprises two nucleic acid sequences encoding the VH-CH and the VL-CL chains.
In a further embodiment, the present invention relates to a host cell containing a nucleic acid sequence according to the present invention or a vector according to the present invention.
In the context of the present invention the term xe2x80x9chost cellxe2x80x9d may be any of a number commonly used in the production of heterologous proteins, including but not limited to bacteria, such as Escherichia coli (Ge et al., 1995), or Bacillus subtilis (Wu et al., 1993), fungi, such as yeasts (Horwitz et al., 1988; Ridder et al., 1995) or filamentous fungus (Nyyssxc3x6nen et al., 1993), plant cells (Hiatt and Ma, 1993; Whitelam et al., 1994), insect cells (Potter et al., 1993; Ward et al., 1995), or mammalian cells (Trill et al., 1995).
In a yet further preferred embodiment, the present invention relates to a modified variant of a wild type bacteriophage coat protein encoded by a nucleic acid sequence according to the present invention, a vector according to the present invention or produced by a host cell according to the present invention.
In another embodiment, the present invention relates to a bacteriophage particle displaying a (poly)peptide/protein on its surface obtainable by a method comprising:
causing or allowing the attachment of said (poly)peptide/protein after expression to a member of the protein coat of said bacteriophage particle, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in said member of the protein coat.
In another embodiment, the present invention relates to a bacteriophage particle displaying a (poly)peptide/protein attached to its surface, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in a member of the protein coat of said bacteriophage particle.
In a preferred embodiment, the bacteriophage particle further contains a vector comprising one or more nucleic acid sequences encoding said (poly)peptide/protein.
In a most preferred embodiment of the present invention, the bacteriophage particle contains a vector according to the present invention, wherein said vector comprises a nucleic acid sequence encoding a modified wild type bacteriophage coat protein and furthermore one or more nucleic acid sequences encoding a (poly)peptide/protein and most preferably comprising at least a functional domain of an immunoglobulin.
The preferred embodiments of the method of the present invention referred to hereinabove mutatis mutandis apply to the bacteriophages of the present invention.
In a further embodiment, the present invention relates to a diverse collection of bacteriophage particles according to the present invention, wherein each of said bacteriophage particles displays a (poly)peptide/protein out of a diverse collection of (poly)peptides/proteins.
A xe2x80x9cdiverse collection of bacteriophage particlesxe2x80x9d may as well be referred to as a xe2x80x9clibraryxe2x80x9d or a xe2x80x9cplurality of bacteriophage particlesxe2x80x9d. Each member of such a library displays a distinct member of the library.
In the context of the present invention the term xe2x80x9cdiverse collectionxe2x80x9d refers to a collection of at least two particles or molecules which differ in at least part of their compositions, properties, and/or sequences. For example, a diverse collection of (poly)peptides/proteins is a set of (poly)peptides/proteins which differ in at least one amino acid position of their sequence. Such a diverse collection of (poly)peptides/proteins can be obtained in a variety of ways, for example by random mutagenesis of at least one codon of a nucleic acid sequence encoding a starting (poly)peptide/protein, by using error-prone PCR to amplify a nucleic acid sequence encoding a starting (poly)peptide/protein, or by using mutator strains as host cells in a method according to the present invention. These and additional or alternative methods for the generation of diverse collections of (poly)peptides/proteins are well-known to one of ordinary skill in the art. A xe2x80x9cdiverse collection of bacteriophage particlesxe2x80x9d may be referred to as a library or a plurality of bacteriophage particles. Each member of such a library displays a distinct member of the library.
In another embodiment, the invention relates to a method for obtaining a (poly)peptide/protein having a desired property comprising:
(a) providing the diverse collection of bacteriophage particles according to the present invention; and
(b) screening said diverse collection and/or selecting from said diverse collection to obtain at least one bacteriophage particle displaying a (poly)peptide/protein having said desired property.
In the context of the present invention the term xe2x80x9cdesired propertyxe2x80x9d refers to a predetermined property which one of the (poly)peptides/proteins out of the diverse collection of (poly)peptides/proteins should have and which forms the basis for screening and/or selecting the diverse collection. Such properties comprise properties such as binding to a target, blocking of a target, activation of a target-mediated reaction, enzymatic activity, and further properties which are known to one of ordinary skill. Depending on the type of desired property, one of ordinary skill will be able to identify format and necessary steps for performing screening and/or selection.
Most preferred is a method, wherein said desired property is binding to a target of interest.
Said target of interest can be presented to said diverse collection of bacteriophage particles in a variety of ways well known to one of ordinary skill, such as coated on surfaces for solid phase biopanning, linked to particles such as magnetic beads for biopanning in solution, or displayed on the surface of cells for whole cell biopanning or biopanning on tissue sections. Bacteriophage particles having bound to said target can be recovered by a variety of methods well known to one of ordinary skill, such as by elution with appropriate buffers, either by using a pH-or salt gradient, or by specific elution using soluble target.
In a preferred embodiment, the method for obtaining a (poly)peptide/protein further comprises:
(ba) contacting said diverse collection of bacteriophage particles with the target of interest;
(bb) eluting bacteriophage particles not binding to the target of interest;
(bc) eluting bacteriophage particles binding to the target of interest by treating the complexes of target of interest and bacteriophages binding to said target of interest formed in step (ba) under reducing conditions.
Under reducing conditions, such as by incubation with DTT, the disulfide bonds are cleaved, thus allowing to recover the specific bacteriophage particles for further rounds of biopanning and/or for identification of the (poly)peptide/proteins specifically binding to said target.